The long-term objectives of this proposal are to analyze the regulatory roles and effects of tumor necrosis factor (TNF) and its two receptors, R1 and R2, in relation to the pathogenesis of and the genetic predisposition to autoimmune diseases in mouse models. The hypothesis of this project is that the (NZBxNZW)F1 mouse model of SLE is characterized by a genetic deficiency in TNF production inherited by the NZW parental strain. We have shown that TNF production is decreased in NZW mice and in a subset of human SLE patients. The deficiency in TNF production in the autoimmune mice may contribute to failure in an apoptotic pathway important in the clonal deletion of autoreactive T and B lymphocytes for the establishment of self-tolerance. We propose that the TNF-induced apoptotic signal can be transduced via a receptor complex between TNF-R2 and Fas antigen. We have developed a large body of preliminary data relating to four aspects of this subject: 1) that low TNF production in NZW mice is linked to MHC; 2) that there is a regulatory defect in the TNF gene in the NZW mouse; 3) that the two TNF receptors are regulated independently, and activate different ion channels; and 4) that TNF-R2 is regulated coordinately with Fas antigen, and they activate similar ion channels. The specific aims of the project are: 1) To provide evidence that the MHC gene contributing to increased susceptibility to lupus in (NZBxNZW)F1 mice is the TNF gene itself. This will be accomplished by analyzing the relationship between disease development, MHC genotype and TNF production under experimental protocols that will control for the inhibitory effects of IL-10. 2) Characterize the regulatory role of 5'-flanking sequences and 3'-untranslated region sequences of the TNFalpha gene in order to provide a molecular basis for low TNF production in NZW mice. 3) To characterize the differences between the two TNF receptors in relation to their expression in lymphocytes; differential activation of ion channels using whole cell clamping and monoclonal antibodies and their ability to induce apoptosis independently, and 4) Determine the role of the TNFalpha and Fas antigen ligands in inducing a TNFR-2/Fas receptor complex, using (a) ligand dependent co-immunoprecipitations and (b) genetic transduction to convert TNF insensitive cell lines into TNF sensitive cells by coexpressing of TNFR-2 and Fas. The experiments described in this proposal concentrate almost exclusively on animal models. The results of these studies should lead to a better understanding of a number of characteristics of autoimmune disease which will lead to new approaches to therapy.